Nucleoside phosphoramidates have a demonstrated utility as pro-drugs of antiviral and anticancer nucleoside monophosphates. In general, there is a requirement that therapeutic nucleosides be converted to at least the corresponding monophosphate before demonstrating biological activity. Nevertheless, many nucleosides are not substrates for the requisite nucleoside kinase. To overcome this hurdle, several approaches have been investigated, including the delivery of nucleoside monophosphoramidates. Although, the nature of the enzyme responsible for phosphoramidate hydrolysis has not been determined, direct evidence of intracellular P—N bond hydrolysis has been demonstrated. Therefore, the identification and characterization of phosphoramidases may facilitate the design of tissue- and species-specific nucleoside phosphoramidates having therapeutic utility.